African trypanosomiasis has two major manifestations: the human disease known as Sleeping Sickness and the animal disease Nagana (meaning loss of spirits in the Zulu language). Animal trypanosomiasis is endemic in equatorial Africa, where it is transmitted, among humans and animals, by Glossina, the Tsetse. The native African fauna are almost universally infected, providing a vast reservoir of potential human pathogens. Focal human epidemics are a serious reality and an increasing threat. If untreated, trypanosomiasis is rapidly fatal. Trypanosomiasis probably kills around 20,000 people annually. The available treatments are unsatisfactory and often ineffective. One of the major reasons for the persistence of African trypanosomes is their unique and deadly efficient mechanism to evade the mammalian immune response. Each trypanosome shields its surface membrane with a replaceable molecular 'coat' composed of a single species of Variant Surface Glycoprotein (VSG). In a process known as Antigenic Variation, the majority of the trypanosome population is destroyed by the immune responses but individual trypanosomes switch coats. Each trypanosome has a repertoire of several hundred VSG genes (vsg), whose expression is tightly regulated by unknown mechanisms. The main objective of our studies is to identify these mechanisms. By developing methods for culturing and genetically manipulating trypanosomes in vitro, we have moved the study of antigenic variation from observation to experimentation. Observations made during the past 10 years suggest several hypotheses for how antigenic variation might be regulated. Now, some of these hypotheses can be experimentally tested. To be expressed, a vsg has to be located in a polycistronic telomeric Expression Site (ES), but this is not sufficient for transcription. There are more than 20 potential ESs, but only one is active at any time. The focus of this proposal is to further characterize Trypanosoma brucei ESs and to identify mechanisms that regulate ES transcription. We will fully sequence one or more ESs. We will try to identify proteins that interact with potential regulatory DNA sequences, including ES promoters and telomeres. We will try to prove which RNA polymerase is responsible for ES transcription. We will explore the potential regulation of ES transcription by trans-acting genes encoded in each ES, or by specific nuclear localization. We will investigate factors that influence the frequency and order of ES switching.